DNA is a locally inflexible polymer. Nonetheless, the intrinsic inflexibility of DNA is somehow overcome in cells, allowing the constant folding and looping of DNA for storage and gene expression. It is fundamentally important to understand how DNA flexibility is enhanced in healthy, and diseased cells. HMGB proteins are abundant non-histone proteins in eukaryotic chromatin. HMGB proteins are thought to function as architectural factors that enhance DNA bending and twisting. We seek to better understand, both the molecular mechanism of HMGB proteins, and the functional role of HMGB proteins in gene expression in living cells. Four specific aims are proposed. 1. Characterization of HMGB mechanism by single molecule experiments. We will test if HMGB proteins cause transient flexible hinges in DNA. 2. Analysis of DNA collapse by cationic protein domains. We will test if HMGB proteins can use cationic domains to cause DNA collapse. 3. Analysis of facilitated DNA looping in bacteria. We will test if HMGB proteins can stabilize bacterial repression loops and substitute for bacterial architectural DNA binding proteins. 4. Analysis of the effect of HMGB proteins on transcription activator position effects in yeast. We will test if HMGB proteins stabilize DNA regulatory loops in yeast. Lav description: The instructions for building living things are coded in recipes (genes) within the cell cookbook (DNA). Very long DNA molecules are difficult to sharply bend and twist, but must nonetheless be folded into many shapes in order to function in cells. By understanding the cell proteins that make DNA flexible, we will learn about tools that may be used to artificially regulate disease genes.